Biosensor and method for quantitating biochemical substrate using the same

ABSTRACT

A biosensor includes: an insulating substrate; an electrode system formed on the insulating substrate which has a working electrode and a counter electrode; and a reaction layer formed on the insulating substrate which contains an oxidoreductase and an electron acceptor. The electron acceptor is ferricinium ion derived from ferrocene electrolyte.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to a biosensor for quantitating abiochemical substrate (specific compound) contained in a sample liquidsuch as whole blood, urine, fruit juice and the like, with accuracy,speed and ease, and a method for quantitating a biochemical substrate bythe biosensor. More particularly, the invention relates to a biosensorfor electrochemically quantitating a concentration of a biochemicalsubstrate in a sample liquid by reacting biochemical substrates such asglucose or cholesterol with an oxidoreductase which can react withspecificity to the biochemical substrates, and a method for quantitatinga biochemical substrate using thereof.

[0003] 2. Description of the Related Art

[0004] Recently, biosensors have been proposed which can easilyquantitate a specific compound (biochemical substrate) in a sampleliquid such as a biological sample or a food without diluting orstirring the sample liquid.

[0005] For example, Japanese Laid-Open Patent Publication No. 3-202764discloses a biosensor including an electrode system formed on aninsulating substrate by screen printing or the like, a reaction layerformed on the electrode system and a space provided as a sample supplypath by using a cover and a spacer. The reaction layer contains ahydrophilic polymer, an oxidoreductase, and an electron acceptor. Such abiosensor can quantitate the concentration of a biochemical substrate ina sample liquid as follows: First, the sample liquid is dropped on thespace in the biosensor so as to be supplied to the reaction layer due tocapillary phenomenon, thereby dissolving the reaction layer. This causesan enzyme reaction between the biochemical substrate in the sampleliquid and the oxidoreductase in the reaction layer, whereby theelectron acceptor in the reaction layer is reduced. After the completionof the enzyme reaction, the reduced electron acceptor iselectrochemically oxidized, whereby the concentration of the biochemicalsubstrate in the sample liquid is quantitated by an oxidation currentvalue.

[0006] In the above-described biosensor, potassium ferricyanide is oftenused as the electron acceptor. Biosensors using potassium ferricyanidehave excellent stability, can be produced at a low cost and thusappropriate in terms of mass production. However, the biosensor usingpotassium ferricyanide as the electron acceptor is associated with asecond-order reaction velocity between the potassium ferricyanide andthe oxidoreductase that is slower than that of a biosensor using anelectron acceptor such as quinone derivatives or another metalliccomplex which is unstable or produced at a high cost. Accordingly, inthe biosensor using potassium ferricyanide, an enzyme reaction takessubstantially long time, thereby causing a problem of not being able topromptly quantitate the concentration of the biochemical substrate.

[0007] Additionally, several biosensors are known which employ ferroceneas an electron acceptor which has a faster second-order reactionvelocity than those of potassium ferricyanide or derivatives thereof.

[0008] Japanese Laid-Open Patent Publication No. 2-240555 discloses aglucose sensor including a working electrode having a photo-curing resinfilm containing a ferrocene compound and a photo-curing resin filmcontaining glucose oxidase sequentially provided on the surface of theworking electrode. Japanese Laid-Open Patent Publication No. 2-99851discloses a glucose sensor including a working electrode having aferrocene compound-containing layer and a glucose oxidase-immobilizedlayer on the ferrocene compound-containing layer on the surface of theworking electrode. In the above-mentioned biosensors, 1,1′-dimethylferrocene, ferrocene, i.e., bis(cyclopentadienyl)iron(II), vinylferrocene or the like is used as the ferrocene compound.

[0009] Japanese Laid-Open Patent Publication No. 5-256812 discloses aglucose sensor including a layer carrying glucose oxidase and aferrocene compound on a working electrode and a means for maintaining apredetermined temperature in the vicinity of the working electrode. Inthis biosensor, ferrocene and/or derivatives thereof is used as theferrocene compound.

[0010] However, any ferrocene compound used in the above-describedglucose sensors normally exists as a reduced form. Therefore, in orderto achieve electron transfer from the biochemical substrate to theworking electrode by an enzyme reaction, the ferrocene compound needs tobe converted into oxidized form on the electrode.

[0011] Japanese Laid-Open Patent Publication No. 6-3316 discloses aglucose sensor (a modified electrode) modified by a hydrophobic redoxsubstance (e.g., a ferrocene) which has been ionized in advance in anaqueous solution, and a hydrophilic enzyme (e.g., glucose oxidase) on asurface of a conductive electrode. The glucose sensor is produced asfollows: Ferrocene is electrolyzed in a phosphate buffer to form asolution containing ferricinium ions. Then, a glucose oxidase is addedto the solution. The resultant mixed solution is applied to orelectrodeposited on the surface of the conductive electrode.

[0012] However, the above-described modified electrode has a problem inthat the phosphate ion and the ferricinium ion form an ion-like complexwhich renders the surface of the electrode inactive. Moreover, a step ofelectrolyzing ferrocene is required in order to produce the modifiedelectrode. As a result, increased production time and increased cost arerequired.

SUMMARY OF THE INVENTION

[0013] According to one aspect of the present invention, a biosensorincludes: an insulating substrate; an electrode system formed on theinsulating substrate which has a working electrode and a counterelectrode; and a reaction layer formed on the insulating substrate whichcontains an oxidoreductase and an electron acceptor. The electronacceptor is ferricinium ion derived from ferrocene electrolyte.

[0014] In one embodiment of the present invention, the ferroceneelectrolyte is selected from the group consisting of ferroceniumhexafluorophosphate and ferrocenium tetrafluoroborate.

[0015] In one embodiment of the present invention, the reaction layerfurther comprises at least one surfactant.

[0016] In one embodiment of the present invention, the reaction layerfurther comprises at least one hydrophilic polymer.

[0017] In one embodiment of the present invention, the oxidoreductase isselected from the group consisting of glucose oxidase; glucosedehydrogenase; lactate oxidase; lactate dehydrogenase; uricase;cholesterol oxidase; a combination of cholesterol oxidase andcholesterol esterase; a combination of glucose oxidase and invertase; acombination of glucose oxidase, invertase and mutarotase; and acombination of fructose dehydrogenase and invertase.

[0018] According to another aspect of the present invention, a method isdisclosed for quantitating the concentration of a biochemical substratein a sample liquid by using a biosensor including an insulatingsubstrate, an electrode system formed on the insulating substrate whichhas a working electrode and a counter electrode and a reaction layerprovided on the insulating substrate which contains an oxidoreductaseand an electron acceptor. The method includes the steps of: adding thesample liquid to the reaction layer; and detecting a response currentvalue by applying a voltage between the working electrode and thecounter electrode. The electron acceptor is ferricinium ion derived fromferrocene electrolyte.

[0019] Thus, the invention described herein makes possible theadvantages of (1) providing a biosensor for promptly quantitating aconcentration of a biochemical substrate with sufficiently short enzymereaction time; (2) providing a biosensor for quantitating aconcentration of a biochemical substrate with accuracy withoutdeteriorating a detecting sensitivity; (3) providing a biosensorproduced at sufficiently low cost; and (4) providing a method forquantitating a concentration of a biochemical substrate using theabove-mentioned biosensor with accuracy and speed.

[0020] These and other advantages of the present invention will becomeapparent to those skilled in the art upon reading and understanding thefollowing detailed description with reference to the accompanyingfigures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]FIG. 1 is an exploded isometric view showing a biosensor accordingto an example of the present invention in which a reaction layer isomitted; and

[0022]FIG. 2 is a schematic cross-sectional view showing a biosensoraccording to an example of the present invention in which a reactionlayer is disposed on an insulating substrate.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] A biosensor according to the present invention includes aninsulating substrate, an electrode system formed on the insulatingsubstrate and a reaction layer disposed on the insulating substrate. Theelectrode system includes a working electrode and a counter electrode.

[0024] The insulating substrate may be a synthetic resin plate made, forexample, of polyethylene terephthalate.

[0025] The electrode system including the working electrode and thecounter electrode may be provided on the insulating substrate by a knownmethod. For example, leads are formed on the insulating substrate. Then,the working electrode and the counter electrode are provided so as to beconnected to the respective leads and insulated from each other. Theleads and the electrodes may be made of any of the known conductivematerials. Examples of the conductive material include carbon, silver,platinum, gold and palladium.

[0026] The reaction layer contains at least one oxidoreductase and atleast one electron acceptor.

[0027] The electron acceptor used in the present invention isferricinium ion derived from ferrocene electrolyte. The term “ferroceneelectrolyte” herein refers to a salt which generates ferricinium ion([(C₅H₅)₂Fe]⁺) upon preparation of a solution with an oxidoreductasewhich will be described later. An anion composing the ferroceneelectrolyte is not specifically limited. Preferred ferroceneelectrolytes are, for example, ferrocenium hexafluorophosphate andferrocenium tetrafluoroborate which are available from Aldrich Inc.

[0028] Although the concentration of the ferrocene electrolyte is notspecifically limited, it is preferably about 1 to about 100 mM in thesample liquid when the reaction layer is dissolved in a sample liquid.When the concentration of the ferrocene electrolyte is smaller than 1 mMin the reaction layer, a measurable range of the concentration of abiochemical substrate may become extremely small. When the concentrationof the ferrocene electrolyte exceeds 100 mM in the reaction layer, thebiosensor may require an increased production cost and may causefluctuation in a response current value and ;poor stability during thestorage since the reaction layer can be broken during the formationthereof.

[0029] The oxidoreductase used in a conventional biosensor such as aglucose sensor, a cholesterol sensor, a lactic acid sensor, a uric acidsensor and a sucrose sensor can be used in the present invention.Examples of oxidoreductase include glucose oxidase (hereinafter,referred to as GOD), glucose dehydrogenase, lactose oxidase, lactosedehydrogenase, uricase, cholesterol oxidase (hereinafter, referred to asChOD), cholesterol esterase (hereinafter, referred to as ChE),invertase, mutarotase and fructose dehydrogenase, and combinationsthereof. In the case where a biosensor according to the presentinvention is a glucose sensor, GOD or glucose dehydrogenase may be usedas the oxidoreductase. In the case where a biosensor according to thepresent invention is a lactic acid sensor, lactose oxidase or lactosedehydrogenase may be used as the oxidoreductase. In the case where abiosensor according to the present invention is a uric acid sensor,uricase may be used as the oxidoreductase. In the case where a biosensoraccording to the present invention is a cholesterol sensor, ChOD or acombination of ChOD and ChE may be used as the oxidoreductase. In thecase where a biosensor according to the present invention is a sucrosesensor, a combination of GOD and invertase; a combination of GOD,invertase and mutarotase; or a combination of fructose dehydrogenase andinvertase may be used as the oxidoreductase.

[0030] The content of the oxidoreductase used in the present inventionis not specifically limited and an appropriate content can be suitablychosen by those skilled in the art. For example, when GOD is used, thecontent of GOD is preferably about 0.1 to about 5 units per biosensor.When a combination of ChOD and ChE is used, the content of ChOD ispreferably about 0.1 to about 5 units per biosensor and the content ofChE is preferably about 0.1 to about 5 units per biosensor. The term “1unit” herein refers to an amount of oxidoreductase required foroxidizing 1 μmol of biochemical substrate to be quantitated in oneminute.

[0031] The reaction layer may further contain at least one ofhydrophilic polymers. Examples of the hydrophilic polymer includecellulose derivatives such as carboxy methyl cellulose (hereinafter,referred to as CMC), hydroxyethyl cellulose, hydroxypropyl cellulose,methyl cellulose, ethyl cellulose, ethyl hydroxy cellulose andcarboxymethyl ethyl cellulose; polyvinyl pyrrolidone; polyvinyl alcohol;gelatin or its derivatives; acrylic acid or its salts; methacrylic acidor its salts; starch or its derivatives; and maleic anhydride or itssalt. In particular, CMC is preferred.

[0032] When the above-mentioned ferricinium ion is employed as theelectron acceptor, ferrocene is produced during sequential enzymereactions. Since the ferrocene generally has low water-solubility, theproduced ferrocene molecules deposit on the reaction layer due to watercontained in a sample liquid. Thus, the reaction layer used in thepresent invention preferably contains at least one of surfactant.Examples of the surfactant include refined lecithin derived fromsoybean, octyl thioglucoside, sodium cholate, dodecyl-β-maltoside,sodium deoxycholic acid, sodium taurodeoxycholate, Triton-X (registeredtrademark), Lubrol PX (registered trademark), DK-ester (registeredtrademark), BIGCHAP (registered trademark) and DeoxyCHAP (registeredtrademark). Specifically, the refined lecithin and the octylthioglucoside are preferred.

[0033] According to the present invention, a lecithin layer containingthe above-mentioned refined lecithin may be further provided on thereaction layer. In the case where the lecithin layer is provided on thereaction layer, a sample liquid can be easily supplied to the reactionlayer.

[0034] Hereinafter, a preferred embodiment of a method for producing abiosensor according to the present invention will be described withreference to FIGS. 1 and 2.

[0035] First, a conductive material such as silver paste is printed onan insulating substrate 1 by screen printing to form leads 2 and 3.Then, another conductive material containing a resin binder is printedon the insulating substrate 1 to form a working electrode 4 which makescontact with the lead 2.

[0036] Then, insulating paste is printed on the insulating substrate 1to form an insulating layer 6. The insulating layer 6 covers theperipheral portion of the working electrode 4, so as to expose a fixedarea of the working electrode 4. As is shown in FIG. 1, the insulatinglayer 6 also covers part of the leads 2 and 3. Around the workingelectrode 4 is formed a ring-shaped counter electrode 5 out of aconductive material containing a resin binder. The counter electrode 5is in contact with the lead 3. In this manner, an electrode system 13including the working electrode 4 and the counter electrode 5 is formedon the insulating substrate 1.

[0037] Alternatively, the biosensor according to the present inventionmay be provided with a three-electrode system including a referenceelectrode (not shown) in addition to the working electrode 4 and thecounter electrode 5 formed on the insulating substrate 1. Thethree-electrode system provides stable response current, thereby furtherstabilizing the measurement accuracy.

[0038] A reaction layer 7 may be formed on the insulating substrate 1 asfollows:

[0039] An aqueous solution containing the hydrophilic polymer is droppedand dried on the electrode system 13 to form a hydrophilic polymerlayer. On the other hand, predetermined amounts of the oxidoreductaseand the ferrocene electrolyte are dissolved in water. Preferably, a fewdrops of surfactant are added to this aqueous solution. Then, theobtained aqueous solution containing the oxidoreductase and theferrocene electrolyte is added dropwise on the hydrophilic polymerlayer. As a result, the hydrophilic polymer is dissolved in the aqueoussolution. Then, the dissolved hydrophilic polymer layer is dried so thatthe reaction layer 7 is formed which is a hydrophilic polymer layerincorporating an oxidoreductase and an electron acceptor. Since theincorporation of the oxidoreductase and the electron acceptor (i.e.,ferricinium ion) into the hydrophilic polymer layer does not requirefurther steps such as stirring, only the hydrophilic polymer exists atthe interface between the reaction layer 7 and the electrode system 13.In other words, since the oxidoreductase and the electron acceptor donot make contact with the surface of the electrode system 13,inactivation of the surface of the electrode system 13 can be avoidedwhich is caused by adsorption of protein on the surface.

[0040] In the case where the hydrophilic polymer layer is not used, anaqueous solution containing oxidoreductase and ferrocene electrolyte isadded dropwise and dried directly on the electrode system 13.

[0041] For repeated application of the biosensor, the oxidoreductase andthe ferrocene electrolyte may be immobilized on the hydrophilic polymerlayer through crosslinking with glutaraldehyde or immobilized on thehydrophilic polymer layer together with a polymeric material such asnitrocellulose or a conventional ion-exchange membrane.

[0042] As shown in FIG. 2, the reaction layer 7 is formed so as to coverthe whole electrode system 13.

[0043] Then, if necessary, a predetermined amount of solution of refinedlecithin in an organic solvent such as toluene is spread and dried onthe reaction layer 7 to form a lecithin layer 8. Finally, as shown inFIG. 1, spacer 10 provided with a sample supply path 12 and a cover 9provided with a hole 11 are disposed in this order above the insulatingsubstrate 1 by a known method. Thus, the biosensor according to thepresent invention is produced.

[0044] A concentration of a biochemical substrate included in a sampleliquid is quantitated by using the biosensor according to the presentinvention, for example, in the following manner.

[0045] First, a sample liquid containing a biochemical substrate isadded to a reaction layer 7 directly or via the sample supply path 12.The reaction layer 7 is dissolved by the sample liquid. After apredetermined period of time, a predetermined level of pulse voltage(e.g., +0.5 V) is anodically applied to the working electrode 4 on thebasis of a voltage at the counter electrode 5. The value of theresultant response current is measured in a known manner. Then, theresponse current value is converted into a concentration value of thebiochemical substrate by using a calibration curve which represents therelationship between the concentration and the response current value ofa biochemical substrate. The calibration curve is obtained in advance bymeasuring known concentrations of the biochemical substrate.

[0046] Hereinafter, a mechanism for obtaining response current valueusing the biosensor according to the present invention will bedescribed.

[0047] For example, in the case where a sample liquid contains glucoseas a biochemical substrate, GOD is used in the reaction layer 7. Whenthe sample liquid is contacted with the reaction layer 7, the reactionlayer 7 is dissolved by the sample liquid and the glucose in the sampleliquid is oxidized by the GOD, thereby producing gluconic lactone. Atthis point, electrons generated through an oxidize reaction of theglucose reduce ferricinium ions existing in the reaction layer 7 intoferrocene. When the above-mentioned pulse voltage is applied to theworking electrode, an oxidation current results which oxidizes theferrocene. The amount of the oxidation current is measured as a reactioncurrent value which is proportional to the concentration of glucoseexisting in the sample liquid.

[0048] In the case where a sample liquid contains cholesterol ester andcholesterol as biochemical substrates, ChE and ChOD are used in thereaction layer 7. When the sample liquid is contacted with the reactionlayer 7, the reaction layer 7 is dissolved by the sample liquid and thecholesterol ester in the sample liquid is converted into cholesterol byChE. Then, all of the cholesterol in the sample liquid is oxidized byChOD, thereby producing cholestenone. At this point, electrons generatedthrough an oxidizing reaction of the cholesterol reduce ferricinium ionsexisting in the reaction layer 7 into ferrocene. When theabove-mentioned pulse voltage is applied to the working electrode, anoxidation current results which oxidizes the ferrocene. The amount ofthe oxidation current is measured as a reaction current value which isproportional to the total concentration of the cholesterol ester andcholesterol existing in the sample liquid.

[0049] Accordingly, the concentration of a biochemical substrateincluded in a sample liquid can be quantitated by the biosensoraccording to the present invention. Furthermore, since ferricinium ionused as an electron acceptor has a second-order reaction velocity fasterthan that of the ferricyanide ion used conventionally, the biosensoraccording to the present invention is capable of quantitating theconcentration of the biochemical substrate in the sample liquid withaccuracy and speed.

[0050] The biosensor according to the present invention can beeffectively used for quantitating the concentration of the biochemicalsubstrate included in biological samples such as whole blood, plasma,serum and urine, materials used in food industry and product thereof(e.g., fruit juice), or the like.

EXAMPLES

[0051] Hereinafter, the present invention will be described by way ofillustrative examples with reference to the accompanying drawings. Thepresent invention, however, is not limited to the following examples. Inthe accompanying drawings, same reference numerals designate samecomponent and the description thereof is partially omitted for the sakeof simplification.

Example 1

[0052] A glucose sensor was produced as follows as an example of abiosensor according to the present invention.

[0053] As shown in FIG. 1, silver paste was printed by screen printingon an insulating substrate 1 made of polyethylene terephthalate to formleads 2 and 3. Then, conductive carbon paste containing a resin binderwas printed on the insulating substrate 1 to form a working electrode 4.The working electrode 4 was formed so as to be in contact with the lead2.

[0054] Next, insulating paste was printed on the insulating substrate 1to form an insulating layer 6. The insulating layer 6 covered theperipheral portion of the working electrode 4, so as to expose a fixedarea of the working electrode 4. Moreover, conductive carbon pastecontaining a resin binder was printed on the insulating substrate 1 toform a ring-shaped counter electrode 5 so that the ring-shaped counterelectrode 5 was in contact with the lead 3.

[0055] Then, an aqueous solution containing GOD and ferroceniumhexafluorophosphate (produced by Aldrich Inc.) was added and dried onthe electrode system 13 (i.e., the working electrode 4 and the counterelectrode 5) to form a reaction layer 7. A toluene solution containinglecithin was added dropwise on the reaction layer 7 to spread over theentire surface of the reaction layer 7 and dried to form a lecithinlayer 8. A spacer 10 and a cover 9 were adhered in this order on thelecithin layer 8, thereby producing the glucose sensor.

[0056] 3 μl of 30 mg/dl aqueous glucose solution was added to theabove-described glucose sensor via a sample supply path 12 in the spacer10. The sample liquid reached as high as the height of the hole 11provided in the cover 9 and dissolved the reaction layer 1. Then, 60seconds after the addition of the sample liquid, a pulse voltage of +0.5V on the basis of a voltage at the counter electrode 5 was anodicallyapplied to the working electrode 4. A response current value wasmeasured 5 seconds after the voltage application.

[0057] Furthermore, response current values were measured with regard toaqueous glucose solutions of 45 mg/dl and 90 mg/dl, respectively in thesame manner as described above by using a fresh glucose sensor for eachmeasurement. The thus-obtained response current values were proportionalto the respective concentrations of the aqueous glucose solutions.

Example 2

[0058] A glucose sensor was produced in the same manner as described inExample 1 except for using ferrocenium tetrafluoroborate (produced byAldrich Inc.) instead of the ferrocenium hexafluorophosphate. By usingthis glucose sensor, response current values were measured for aqueousglucose solutions having the same concentrations as those described inExample 1 in the same manner as described in Example 1. The obtainedresponse current values were proportional to the respectiveconcentrations of the aqueous glucose solutions.

Example 3

[0059] An electrode system 13 was formed on an insulating substrate 1 inthe same manner as described in the Example 1.

[0060] Then, an aqueous solution containing 0.5% by weight of CMC wasadded dropwise on the electrode system 13 (i.e., working electrode 4 andthe counter electrode 5) and dried to form a CMC layer. An aqueoussolution containing GOD and ferrocenium hexafluorophosphate was addeddropwise and dried on the CMC layer to form a reaction layer 7.Furthermore, a toluene solution containing lecithin was added dropwiseon the reaction layer 7 to spread over the entire surface of thereaction layer 7 and dried to form a lecithin layer 8. A spacer 10 and acover 9 were adhered in this order on the lecithin layer 8, therebyproducing the glucose sensor.

[0061] 3 μl of 30 mg/dl aqueous glucose solution was added as a sampleliquid to the above-described glucose sensor via a sample supply path 12in the spacer 10. The sample liquid reached as high as a height of ahole 11 provided in the cover 9 and dissolved the reaction layer 7.Then, 60 seconds after the addition of the sample liquid, a pulsevoltage of +0.5 V on the basis of a voltage at the counter electrode 5was anodically applied to the working electrode 4. A response currentvalue was measured 5 seconds after the voltage application.

[0062] Furthermore, response current values were measured with regard toglucose aqueous solutions of 45 mg/dl and 90 mg/dl, respectively, in thesame manner as described above by using a fresh glucose sensor for eachmeasurement. The thus-obtained response current values were proportionalto the respective concentrations of the aqueous glucose solutions.

Example 4

[0063] A glucose sensor was produced in the same manner as described inExample 3 except for using ferrocenium tetrafluoroborate instead of theferrocenium hexafluorophosphate. By using this glucose sensor, responsecurrent values were measured in the same manner as described in Example3 for aqueous glucose solutions having the same concentrations as thosedescribed in Example 3. The obtained response current values wereproportional to the respective concentrations of the aqueous glucosesolutions.

Example 5

[0064] A glucose sensor was produced in the same manner as described inExample 3 except that refined lecithin derived from soybean (produced bySIGMA Chemical Co.) was added as a surfactant to the aqueous solutioncontaining GOD and ferrocenium hexafluorophosphate. This aqueoussolution was added dropwise on the CMC layer. By using this glucosesensor, response current values were measured in the same manner asdescribed in Example 3 for aqueous glucose solutions having the sameconcentrations as those described in Example 3. The obtained responsecurrent values were proportional to the respective concentrations of theaqueous glucose solutions.

Example 6

[0065] A glucose sensor was produced in the same manner as described inExample 3 except that ferrocenium tetrafluoroborate was used instead offerrocenium hexafluorophosphate and that refined lecithin derived fromsoybean was added as a surfactant to the aqueous solution containing GODand ferrocenium tetrafluoroborate. This aqueous solution was addeddropwise on the CMC layer. By using this glucose sensor, responsecurrent values were measured in the same manner as described in Example3 for aqueous glucose solutions having the same concentrations as thosedescribed in Example 3. The obtained response current values wereproportional to the respective concentrations of the aqueous glucosesolution.

Example 7

[0066] An electrode system 13 was formed on an insulating substrate 1 inthe same manner as described in Example 1.

[0067] Then, an aqueous solution containing 0.5% by weight of CMC wasadded dropwise and dried on the electrode system 13 (i.e., the workingelectrode 4 and the counter electrode 5) to form a CMC layer. Octylthioglucoside was added as surfactant to an aqueous solution containingChE, ChOD and ferrocenium hexafluorophosphate. The obtained aqueoussolution was added dropwise and dried on the CMC layer to form areaction layer 7. Furthermore, the toluene solution containing lecithinwas added dropwise on the reaction layer 7 to spread over the entiresurface of the reaction layer 7 and dried to form a lecithin layer 8. Aspacer 10 and a cover 9 were adhered in this order on the lecithin layer8, thereby producing the cholesterol sensor.

[0068] 3 μl of standard solution containing 50 mg/dl of cholesterol and150 mg/dl of cholesterol ester was added as a sample liquid to theabove-described cholesterol sensor via a sample supply path 12 in thespacer 10. The sample liquid reached as high as a height of a hole 11provided in the cover 9 and dissolved the reaction layer 7. Then, 180seconds after the addition of the sample liquid, a pulse voltage of +0.5V on the basis of a voltage at the counter electrode 5 of the electrodesystem 13 was anodically applied to the working electrode 4. A responsecurrent value corresponding to the total concentration of thecholesterol ester and cholesterol was measured 5 seconds after thevoltage application.

[0069] Furthermore, response current values were measured with regard toa standard solution containing 300 mg/dl of cholesterol ester and 100mg/dl of cholesterol and a standard solution containing 450 mg/dl ofcholesterol ester and 150 mg/dl of cholesterol, respectively in the samemanner as described above by using a fresh cholesterol sensor for eachmeasurement. The thus-obtained response current values were proportionalto the respective total concentrations of the cholesterol ester andcholesterol existing in the sample liquids.

Example 8

[0070] A cholesterol sensor was produced in the same manner as describedin Example 7 except that ChE was not contained in the reaction layer 7.By using this cholesterol sensor, reaction current values were measuredfor the same standard solutions containing cholesterol ester andcholesterol, respectively. The thus-obtained response current valueswere only proportional to the respective concentrations of cholesterolin the standard solutions.

Example 9

[0071] A cholesterol sensor was produced in the same manner as describedin Example 7 except for using ferrocenium tetrafluoroborate instead ofthe ferrocenium hexafluorophosphate. By using this cholesterol sensor,response current values were measured in the same manner as described inExample 7 for standard solutions containing cholesterol ester andcholesterol of the same concentrations as those described in Example 7.The obtained response current values were proportional to the respectivetotal concentrations of cholesterol ester and cholesterol in the sampleliquids.

Example 10

[0072] A cholesterol sensor was produced in the same manner as describedin Example 7 except that ChE was not contained in the reaction layer 7and ferrocenium tetrafluoroborate was used instead of the ferroceniumhexafluorophosphate. By using this cholesterol sensor, reaction currentvalues were measured for the same standard solutions containingcholesterol ester and cholesterol, respectively. The thus-obtainedresponse current values were only proportional to the respectiveconcentrations of cholesterol in the standard solutions.

[0073] Various other modifications will be apparent to and can bereadily made by those skilled in the art without departing from thescope and spirit of this invention. Accordingly, it is not intended thatthe scope of the claims appended hereto be limited to the description asset forth herein, but rather that the claims be broadly construed.

What is claimed is:
 1. A biosensor comprising: an insulating substrate;an electrode system formed on the insulating substrate which includes aworking electrode and a counter electrode; and a reaction layer formedon the insulating substrate which contains an oxidoreductase and anelectron acceptor, wherein the electron acceptor is ferricinium ionderived from ferrocene electrolyte.
 2. A biosensor according to claim 1, wherein the ferrocene electrolyte is selected from the groupconsisting of ferrocenium hexafluorophosphate and ferroceniumtetrafluoroborate.
 3. A biosensor according to claim 1 , wherein thereaction layer further comprises at least one surfactant.
 4. A biosensoraccording to claim 1 , wherein the reaction layer further comprises atleast one hydrophilic polymer.
 5. A biosensor according to claim 1 ,wherein the oxidoreductase is selected from the group consisting ofglucose oxidase; glucose dehydrogenase; lactate oxidase; lactatedehydrogenase; uricase; cholesterol oxidase; a combination ofcholesterol oxidase and cholesterol esterase; a combination of glucoseoxidase and invertase; a combination of glucose oxidase, invertase andmutarotase; and a combination of fructose dehydrogenase and invertase.6. A method for quantitating the concentration of a biochemicalsubstrate in a sample liquid by using a biosensor including aninsulating substrate, an electrode system formed on the insulatingsubstrate which has a working electrode and a counter electrode and areaction layer provided on the insulating substrate which contains anoxidoreductase and an electron acceptor, comprising the steps of: addingthe sample liquid to the reaction layer; and detecting a responsecurrent value by applying a voltage between the working electrode andthe counter electrode, wherein the electron acceptor is ferricinium ionderived from ferrocene electrolyte.
 7. A method according to claim 6 ,wherein the ferrocene electrolyte is selected from the group consistingof ferrocenium hexafluorophosphate and ferrocenium tetrafluoroborate. 8.A method according to claim 6 , wherein the reaction layer furthercomprises at least one surfactant.
 9. A method according to claim 6 ,wherein the reaction layer further comprises at least one hydrophilicpolymer.
 10. A method according to claim 6 , wherein the oxidoreductaseis selected from the group consisting of glucose oxidase; glucosedehydrogenase; lactate oxidase; lactate dehydrogenase; uricase;cholesterol oxidase; a combination of cholesterol oxidase andcholesterol esterase; a combination of glucose oxidase and invertase; acombination of glucose oxidase, invertase and mutarotase; and acombination of fructose dehydrogenase and invertase.
 11. A methodaccording to claim 6 , wherein the biochemical substrate is selected thegroup consisting of glucose, cholesterol, lactic acid, uric acid andsucrose.